Patent Application
20230017359
The DNA alkylating anti-cancer prodrug targeting overexpressed aldo-keto reductase 1C3 (AKR1C3) developed by our company (see Patent Applications:
DNA alkylating agents, PCT Patent Application No. PCT/US2016/021581 (Publication No. WO2016/145092A), which corresponds to Chinese Patent Application No. 2016800150788 (Publication No. CN107530556A);
(R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N′-bis (ethylidene)phosphoramidate, compositions and methods of use and preparation thereof, a S configuration compound in PCT Patent Application No. PCT/US2016/062114 (Publication No. WO2017087428A1), which corresponds to Chinese Patent Application No. 2016800446081 (Publication No. CN108290911A);
Nitrobenzyl derivative anti-cancer agents, PCT Patent Application No. PCT/US2016/025665 (Publication No. WO2016/161342), which corresponds to Chinese Patent Application No. 2016800200132 (Publication No. CN108136214A)) is a DNA alkylating anti-cancer prodrug targeting aldo-keto reductase 1C3 (AKR1C3). Compound A in the form of prodrug (i.e., a compound of the general formula I claimed by the invention) is subjected to single-electron reduction under the catalysis of AKR1C3 enzyme in a biochemical environment in cells to obtain an intermediate B which is also unstable and then further reduced under the action of AKR1C3 enzyme to obtain three intermediates C, which are still unstable and are subjected to 1,4 elimination reaction to finally obtain
and cytotoxic H-L1-D, which plays a role in killing cancer cells, as shown in
Specifically, the compound with a name of (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N′-bis (ethylidene)phosphoramidate, also known as a S configuration compound of OBI-3424 and TH-2870 (CAS No. 2097713-69-2 has the following structure:
A Phase I clinical trial has been conducted in the United States and China, respectively.
According to Document 3 and the mechanism of drug, it is known that the drug is only effective for patients with AKR1C3 expression, so it is necessary to detect the expression level of AKR1C3 in patients.
The conventional detection method is to directly obtain tumor tissue sections from the patients (Document 4: Christopher P. Guise, Maria R. Abbattista, Rachelle S. Singleton, et al, The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3, Cancer Res, 70 (4): 1573-1584; DOI:10.1158/0008-5472.CAN-09-3237), and then use an immunohistochemical staining method (IHC) for detection.
However, tumor tissue sections cannot be obtained under certain occasions and conditions: tumor tissue sections of certain patients are lost so that they cannot be or are inconvenient to be obtained again (tumor tissue sections of certain patients are lost due to improper storage); certain patients are unwilling to accept the section sampling operation (the sections are obtained from the body part of the patient by forceps, excision or puncture aspiration and the like according to different situations) due to culture, concept or religious habit and the like; the patients with certain cancers or tumors cannot be sectioned.
The invention provides a novel detection method which can be adapted to a plurality of biological samples to detect the expression level of associated AKR1C3.
The research and development group conceived that since the compounds disclosed in the above three applications for invention serve as specific substrates of aldo-keto reductase AKR1C3 (hereinafter abbreviated as specific substrates), which are specifically activated by the aldo-keto reductase AKR1C3 and metabolized to obtain cytotoxic alkylating agents, that is, the aldo-keto reductase AKR1C3 needs to bind to the specific substrate to function.
According to research documents (Document 5: Samad T A, Moore K A, Sapirstein A, et al. Interleukin-1[beta]-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity[J]. Nature, 2001, 410(6827):471-5;
The inventors envisaged that since AKR1C3 plays a role in the conversion of prostaglandins, whether the content change of prostaglandin F2 (a product of the conversion process) or prostaglandin H2/D2 (a reactant of the conversion process), can be used to associate with expression level of AKR1C3. Specifically, whether there are the following corresponding relationships:
Before and after administration of the inhibitor or agonist of AKR1C3, whether the corresponding content change of product prostaglandin F2 before and after the conversion process or reactant prostaglandin H2/D2 in the conversion process indicates that a large change means that the expression level of AKR1C3 is high (as shown in
The rationality of the above idea can be verified through in vivo animal experiments, and the above guess and reasoning can be verified through analysis of experimental results.
The following technical schemes are provided for this purpose.
Scheme 1: A method for measuring expression level of AKR1C3 enzyme
The present invention provides a method for measuring the expression level of AKR1C3 enzyme, comprising measuring the content of PGF2α and/or PGD2 and/or PGH2 to associate with the expression level of AKR1C3 enzyme in a biological sample.
The expression level of AKR1C3 enzyme is generally evaluated by non-expression (undetectable), low, medium and high. For example, according to the description in Document 4, the grading is determined according to the degree of staining after staining using IHC method: diffuse, determined to be highly expressed when all cells are stained; moderate, determined to be mediumly expressed when most or many cells are stained; few, determined to be lowly expressed when only a few cells are stained; and non-expression, determined to be negative when staining was not observed.
Of course, high, medium and low grades can also be determined according to the percentage of the stained area to the overall area after the sample is subjected to IHC staining.
In this scheme, the determination is made directly by measuring the content of PGF2α and/or PGD2 and/or PGH2:
A large number of sample statistics were subjected to generate an association formula between the content of PGF2α and/or PGD2 and/or PGH2 in the population and the expression level of AKR1C3 enzyme (the expression level herein is quantified by the percentage of the stained area to the overall area after IHC staining). That is, when the content of PGF2α and/or PGD2 and/or PGH2 in a person is greater than the high value H, the expression level of AKR1C3 enzyme could be directly considered to be high within a certain confidence range; and when the content of PGF2α and/or PGD2 and/or PGH2 in a person is lower than the low value L, the expression level of AKR1C3 enzyme could be directly considered to be low within a certain confidence range, while the remaining ones between the high value H and the low value L are determined as medium expression.
Further, in the above scheme, the association refers to:
when the content of PGF2α is in the range of A1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content of PGF2α is in the range of B1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content of PGF2α is in the range of C1, the expression level of AKR1C3 enzyme in the biological sample is low;
and/or
when the content of PGD2 and/or PGH2 is in the range of A2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content of PGD2 and/or PGH2 is in the range of B2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when that content of PGD2 and/or PGH2 is in the range of C2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of A1 is greater than or equal to the maximum value in the range of B1, the minimum value in the range of B1 is greater than or equal to the maximum value in the range of C1, i.e., the range of A1 is a (m, n) interval, the range of B1 is a (p, q) interval, and the range of C1 is a (r, s) interval, then the minimum value n in the range of A1 is greater than or equal to the maximum value p in the range of B1, and the minimum value q in the range of B1 is greater than or equal to the maximum value r in the range of C1. The units for the six ranges A1, B1, C1, and A2, B2, C2 are the units of content (mass/volume) and pg/ml in human samples.
The minimum value in the range of A2 is greater than or equal to the maximum value in the range of B2, and the minimum value in the range of B2 is greater than or equal to the maximum value in the range of C2.
Obviously, the above direct determination method is the most rapid and convenient.
Further, considering that prostaglandin F2 in roadmap 1/2/3 is the final product of the metabolic pathway, and its content is directly related to AKR1C3, while the metabolism of prostaglandin D2/H2 is also affected by other factors (such as there are other metabolic pathways), therefore, the expression level of AKR1C3 is not the only factor for determining prostaglandin D2/H2. Therefore, the method for measuring the content of prostaglandin F2 is the preferred one among the above three schemes.
Since it is necessary to determine six ranges: A1, B1, C1, and A2, B2, C2, respectively, which are obtained by smoke statistics of a population, there may be a problem that a conclusion of a statistical sample may not necessarily represent a certain specific subject: in fact, the case that a certain sample just falls outside the statistical sample cannot be ruled out, and the statistic and association of a large number of samples are also a time-consuming and resource-consuming work, and more importantly, differences in race and living region may render that the result of the method is inaccurate or applicable population is narrow.
To this end, this scheme further proposes a method using drugs for interference: a method for measuring the expression level of AKR1C3 enzyme, comprising measuring the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interference drug to associate with the expression level of AKR1C3 enzyme in a biological sample.
The above method comprises associate with the expression level of AKR1C3 enzyme by obtaining the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interference drug. Similarly, a large number of sample statistics are subjected to generate a association formula between the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of the same interference drug in the population and the expression level of AKR1C3 enzyme (the expression level herein is quantified by the percentage of the stained area to the overall area after IHC staining), that is, when the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of a certain interference drug in a person is greater than the high value H, the expression level of AKR1C3 enzyme can be directly considered as high within a certain confidence range; and when the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of a certain interference drug in a person is lower than the low value L, the expression level of AKR1C3 enzyme can be directly considered as low within a certain confidence range, while the remaining ones between the high value H and the low value L are determined as medium expression.
Based on this method, different datas of different interference drugs (different AKR1C3 inhibitors and different AKR1C3 agonists) can be obtained statistically to verify each other, so that the influence caused by the difference of race and living region can be reduced or weakened to a certain extent, and conclusions obtained from smaller samples can be adapted to a wider population.
Further, in the above scheme, the association refers to:
when the content change of PGF2α is in the range of α1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content change of PGF2α and/or PGD2 and/or PGH2 is in the range of b1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change of PGF2α and/or PGD2 and/or PGH2 is in the range of c1, the expression level of AKR1C3 enzyme in the biological sample is low;
and/or
when the content change of PGD2 and/or PGH2 is in the range of a2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content change of PGD2 and/or PGH2 is in the range of b2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change of PGD2 and/or PGH2 is in the range of c2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of a1 is greater than or equal to the maximum value in the range of b1, and the minimum value in the range of b1 is greater than or equal to the maximum value in the range of c1, i.e.
the minimum value in the range of a2 is greater than or equal to the maximum value in the range of b2, and the minimum value in the range of b2 is greater than or equal to the maximum value in the range of c2.
Further, considering that prostaglandin F2 in roadmap 1/2/3 is the final product of the metabolic pathway, and its content is directly related to AKR1C3, while the metabolism of prostaglandin D2/H2 is also affected by other factors (such as there are other metabolic pathways), therefore, the expression level of AKR1C3 is not the only factor for determining prostaglandin D2/H2. Therefore, the method for measuring the content change of prostaglandin F2 before and after administration of the interference drug is the preferred one among the above three schemes.
Furthermore, in the above method for performing association using absolute change, there may be a case where the absolute value of the content change of PGF2α and/or PGD2 and/or PGH2 in the statistical sample is large, so that a certain value in the statistical sample is too far from the center value (high, medium, and low expressions each correspond to three center values), and thus the change rate is introduced to overcome this problem.
The present invention provides a method for measuring the expression level of AKR1C3 enzyme, comprising measuring the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after application of the interference drug to associate with the expression level of AKR1C3 enzyme in the biological sample. Similarly, a large number of sample statistics are subjected to generate an association formula between the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of the same interference drug in the population (defined as the percentage of the content change to the content before drug application) and the expression level of AKR1C3 enzyme (the expression level herein is quantified by the percentage of the stained area to the overall area after IHC staining), that is, when the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of a certain interference drug in a person is greater than the high value H, the expression level of AKR1C3 enzyme can be directly considered as high within a certain confidence range; and when the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of a certain interference drug in a person is lower than the low value L, the expression level of AKR1C3 enzyme can be directly considered as low within a certain confidence range, while the remaining ones between the high value H and the low value L are determined as medium expression.
Based on the method, different datas of different interference drugs (different AKR1C3 inhibitors and different AKR1C3 agonists) can be obtained statistically to verify each other, and the concept of change rate is introduced, so that the effect of the absolute content of PGF2α and/or PGD2 and/or PGH2 in an individual is weakened to a certain extent, and the association between the change rate and the expression level of AKR1C3 is highlighted; therefore, the influence caused by difference of race and living region can be better alleviated and weakened; and conclusions obtained by sample statistical analysis can be applied to a wider population and have a more accurate association result.
Furthermore, in the above scheme, the association refers to:
when the content change rate of PGF2α is in the range of α1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content change rate of PGF2α is in the range of β1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change rate of PGF2α is in the range of γ1, the expression level of AKR1C3 enzyme in the biological sample is low,
and/or
when the content change rate of PGD2 and/or PGH2 is in the range of a2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content change rate of PGD2 and/or PGH2 is in the range of β2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change rate of PGD2 and/or PGH2 is in the range of γ2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of α1 is greater than or equal to the maximum value in the range of β1, and the minimum value in the range of β1 is greater than or equal to the maximum value in the range of γ1,
the minimum value in the range of α2 is greater than or equal to the maximum value in the range of β2, and the minimum in the range of β2 is greater than or equal to the maximum value in the range of γ2.
Further, considering that prostaglandin F2 in roadmap 1/2/3 is the final product of the metabolic pathway, and its content is directly related to AKR1C3, while the metabolism of prostaglandin D2/H2 is also affected by other factors (such as there are other metabolic pathways), therefore, the expression level of AKR1C3 is not the only factor for determining prostaglandin D2/H2. Therefore, the method for measuring the content change rate of prostaglandin F2 before and after administration of the interference drug is the preferred one among the above three schemes.
Further, the interference drug is an AKR1C3 enzyme inhibitor or an AKR1C3 enzyme agonist.
Known AKR1C3 enzyme inhibitors include those disclosed in the following Patent Applications:
and indomethacin and other Chinese herbal medicines, of course, TH-2870 and the like.
Chemical Structural Formula of TH2870
An agonist is a chemical that binds to and activates a receptor to produce a biological reaction. Agonists cause the effect, while antagonists block the effect of the agonist, and inverse agonists cause the effect opposite to the agonist.
Preferably, the AKR1C3 enzyme inhibitor is
Among the above preferred compounds,
and the isomers thereof in R/S configuration are compounds with AKR1C3 inhibitory activity verified by experiments, while indomethacin is a drug that has been marketed.
Those skilled in the art can select different forms of biological samples according to the situation, including blood or serum, tissues or skin or tumor tissue sections, and perform detection according to different situations. In contrast, since blood is readily available, it is preferable that the biological sample (biological specimen) is blood.
There are relatively mature methods for detecting the content of prostaglandins (PGF2α and/or PGD2 and/or PGH2) in blood. For example, there are commercial kits and related methods for enzyme-linked immunosorbent assay (ELISA) kits for detecting PGF2α.
The present invention provides a measuring method, comprising the following operations:
Operation I. Measurement of the content of PGF2a and/or PGD2 and/or PGH2 in a living body, a living biological organ or a living biological tissue.
Obviously, if we have a reliable (with high enough confidence level) statistical data conclusion that matches the living region and race corresponding to the measured living body or living biological organ or living biological tissue subject, we can obtain the expression level of AKR1C3 corresponding to the living body or organ or tissue according to the corresponding association relationship through Operation I.
If there are no suitable statistical data conclusions, the following interference operations are required:
Operation II. Administration of the interference drug to the living body, the living biological organ or the living biological tissue.
The administration of the interference drug herein needs to be adjusted according to different situations: the living body (animal or human) is administered with the interference drug: AKR1C3 inhibitors or agonists by oral or injection administration (preferably oral administration, with a faster effect); for the living biological organ, either infusion or injection administration can be performed, and the living biological tissue can only be immersed in a solution of AKR1C3 inhibitor or agonist (containing appropriate buffer components) for incubation reaction.
Of course, different administration methods have different optimal measurement times: in general, the amount of drug to be applied or the concentration of exposure time (for tissues subjected to immersion incubation) in different living bodies or different organs and tissues thereof should be experimentally explored to obtain the optimal amount of drug to be applied or the optimal exposure time as well as the time for sampling and testing again after administration.
Operation III. Measurement of the content of PGF2α and/or PGD2 and/or PGH2 in the living body, the living biological organ or the living biological tissue after administration of the interference drug.
Obviously, the specific time “after administration of the interference drug” needs to be experimentally explored, which is related to the half-life T1/2 of the interference drug for administration to the living body.
Operation IV. Calculation of the content change and the content change rate before and after administration of the interference drug, and obtaining the expression level of the AKR1C3 enzyme in the biological sample according to the corresponding relationship.
Scheme 2: A Method for Screening and Administering
A method for screening and administering to a patient with cancer, tumor, or a disorder caused by cancer or tumor, or a cell proliferative disease comprises administering an AKR1C3 enzyme-activated anti-cancer prodrug to the patient after the expression level of AKR1C3 enzyme is obtained by using the above method for measuring the expression level of AKR1C3 enzyme.
In the present invention, the AKR1C3 enzyme-activated anti-cancer prodrugs, i.e., compounds in the form of prodrugs, are activated by catalysis of AKR1C3 enzymes in the biochemical environment in cells to finally obtain a cytotoxic toxin to play a role in killing cancer cell. See the compounds disclosed in the following patent applications:
The compounds of the general formula and specific compounds disclosed in the above patent applications all belong to AKR1C3-activated anti-cancer prodrugs (compounds), and the above applications are herein incorporated into the description of the present application.
A method for screening and administering to a patient with cancer, tumor, or a disorder caused by cancer or tumor, or a cell proliferative disease comprises administering an AKR1C3 enzyme-activated anti-cancer prodrug to the patient when the level of enzyme is high after the expression level of AKR1C3 enzyme is obtained from using the above method for measuring the expression level of AKR1C3 enzyme.
A method for screening and administering to a patient with cancer, tumor, or a disorder caused by cancer or tumor, or a cell proliferative disease comprises administering an AKR1C3 enzyme-activated anti-cancer prodrug to the patient when the enzyme level is high or medium after the expression level of AKR1C3 enzyme is obtained from using the above method for measuring the expression level of AKR1C3 enzyme.
Further, in the above administration method, according to the compounds disclosed in Patent Application No. PCT/US20167021581 (Publication No. WO2016145092A1), which corresponds to the Chinese Patent Application No. 2016800150788 (Publication No. CN107530556A), the AKR1C3 enzyme-activated anti-cancer prodrug comprises the compound of structural formula I:
wherein,
X10 is O, S, SO or SO2;
A is C6-C10 aryl or substituted aryl, 5-15 membered heteroaryl or substituted heteroaryl or —N═CR1R2; wherein R1 and R2 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
R13 and R14 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether, or the R13 and R14 groups together with the nitrogen atom to which they are bonded form 5-7 membered heterocyclyl;
T comprises an amino phosphate alkylating agent, i.e., T is -L-D, and includes the following six cases:
-D is —P(Z1)(Z5—X5—Y5)n, Z1 is O or S, Z5 is N, S or O, X5 is an optionally substituted ethylidene, Y5 is a halogen atom or —OSO2—R20, R20 is an optionally substituted hydrocarbyl, aryl, cycloalkyl, heterocyclyl or heteroaryl, n is 1 or 2, and L is selected from —O—, —S—, —OCOO—, —NR6CO—, —OCO—, —NR6SO2—, —OCONR6—, quaternary ammonium, sulfonate group-OSO2—;
or
Z1 is O or S, and Z5—X5—Y5 is an aziridinyl —NCH2CH2 moiety;
or
-L- is —O—, -D is —P(Z1)(Z5—X5—Y5)n, Z1 is O or S, Z5 is N, S or O, X5 is an optionally substituted ethylidene, Y5 is a halogen atom or —OSO2—R20, R20 is an optionally substituted hydrocarbyl, aryl, cycloalkyl, heterocyclyl or heteroaryl, and n is 1 or 2;
or
-L- is —O—, Z1 is O or S, and Z5—X5—Y5 is an aziridinyl —NCH2CH2 moiety;
or
-L-D is —OP(Z1)(NR30CH2CH2Cl)2, —OP(Z1)(NR30CH2CH2Br)2, —OP(Z1)(NR302)(N(CH2CH2X)2), —OP(Z1)(N(CH2)2)2, or —OP(Z1)(N(CH2CH2Cl)2)2, wherein each R30 is independently H, C1-C6 hydrocarbyl or two R30 groups together with the nitrogen atom to which they are linked form 5-7 membered heterocycle, Z1 is O or S, and X1 is Cl, Br or —OSO2Me;
or
-L-D is —OP(Z1)(NHCH2CH2Cl)2, —OP(Z1)(NHCH2CH2Br)2, —OP(Z1)(NH2)(N(CH2CH2X)2), —OP(Z1)(N(CH2)2)2 or —OP(Z1)(N(CH2CH2Cl)2)2, and X1 is Cl, Br, or —OSO2Me;
and the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl and the ether group are substituted or unsubstituted.
The substituent is a halogen atom, cyano or isocyano, thiocyano or isothiocyano, hydroxyl, mercapto, amino, oximido, a hydrazone group, OTs, OMS, C1-C3 alkyl or substituted alkyl, C1-C3 alkoxy or substituted alkoxy, C2-C3 alkenyl or substituted alkenyl, C2-C3 alkynyl or substituted alkynyl, C3-C8 cycloalkyl or substituted cycloalkyl, an aromatic ring, heterocycle, a heteroaromatic ring and a fused ring or a substituted aromatic ring, heterocycle, a heteroaromatic ring and a fused ring, wherein the substituted mode is monosubstituted or geminal disubstituted, and the substituent is a halogen atom, cyano or isocyano, thiocyano or isothiocyano, hydroxyl, mercapto, amino, oximido, a hydrazone group, OTs or OMs.
Further, the AKR1C3 enzyme-activated anti-cancer prodrug contains a compound having a structural formula selected from the group consisting of:
According to Patent Application No. PCT/US2016/025665 (Publication No. WO2016161342), which corresponds to Chinese Patent Application No. 2016800446081 (Publication No. CN108290911A), the above compound of structural formula I further comprises:
Further, in the above administration method, according to Patent Application No. PCT/US2016/062114 (Publication No. WO2017087428), which corresponds to Chinese Patent Application No. 2016800200132 (Publication No. CN108136214A), the AKR1C3 enzyme-activated anti-cancer prodrug contains a compound of structural formula II:
wherein
X10 is O, S, SO or SO2,
A is C6-C10 aryl or substituted aryl, 5-15 membered heteroaryl or substituted heteroaryl or —N═CR1R2; wherein R1 and R2 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
each R is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR13R14 or —NR13COR14;
R13 and R14 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether, or R13 and R14 together with the nitrogen atom to which they are bonded form 5-7 membered heterocyclyl;
L1 and D are as defined in the description, and the specific definitions are as follows:
L1 is selected from:
wherein R40 and R41 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle or 5-15 membered heteroaryl;
R42 is a C2-C3 alkylene or heteroalkylene optionally substituted with 1-3 C1-C6 alkyl; V(−) is any anion, preferably a pharmaceutically acceptable anion; and
D is a moiety that makes D-OH an anti-cancer drug, wherein OH is aliphatic hydroxyl or phenolic hydroxyl; in other words, D is a group after hydroxyl of the anti-cancer drug D-OH is removed;
or
L1 is:
wherein R40 is as defined above, R43 is hydrogen or together with D forms heterocycle and the phenyl moiety is optionally substituted; and
D is a moiety that makes D-NR43H an anti-cancer drug; in other words, D is a group after amino or amine of the anti-cancer drug D-NR43H is removed;
or
L1 is a bond, —O—C(R40R41)—, —O—C(R40R41)—NR40R41(+)—C(R40R41)— or
wherein R40, R41 and V are as defined above; and
D is an anti-cancer drug containing a primary or secondary amine, wherein the primary or secondary amine is bonded to L1; and
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl and the ether group are substituted or unsubstituted.
The substituent is a halogen atom, cyano or isocyano, thiocyano or isothiocyano, hydroxyl, mercapto, amino, oximido, a hydrazone group, OTs, OMS, C1-C3 alkyl or substituted alkyl, C1-C3 alkoxy or substituted alkoxy, C2-C3 alkenyl or substituted alkenyl, C2-C3 alkynyl or substituted alkynyl, C3-C8 cycloalkyl or substituted cycloalkyl, an aromatic ring, heterocycle, a heteroaromatic ring and a fused ring or a substituted aromatic ring, heterocycle, a heteroaromatic ring and a fused ring, wherein the substituted mode is monosubstituted or geminal disubstituted, and the substituent is a halogen atom, cyano or isocyano, thiocyano or isothiocyano, hydroxyl, mercapto, amino, oximido, a hydrazone group, OTs or OMs.
Further, in the above administration method, in the compound of structural formula II contained in the AKR1C3 enzyme-activated anti-cancer prodrug,
D-OH is selected from the following anti-cancer drugs containing an —OH group: gemcitabine, estramusting, pudnimnstine, chlorozotocin, ranimustine, mannomustine, mitobronitol, dibromodulcitol, aclacinomycins, anthramycin, bleomycin, carubicin, carzinophilin, chromomycin, actinomycin D, daunorubicin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, fludarabine, ancitabine, azacitidine, 6-azauridine, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, L-asparaginase, pulmozyme, aceglatone, elliptinium acetate, etoglucid, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoxantrone, mopidamol, pentostatin, pirarubicin, podophyllinic acid, sizofiran, paclitaxel, teniposide, tenuazonic acid, vinblastine and vincristine;
NR43H is selected from the following anti-cancer drugs: erlotinib, meturedepa, uredepa, imatinib, trimethylolomelamine, gefitinib, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, actinomycin, anthramycin, bleomycin, actinomycin C, carubicin, carzinophilin, actinomycin D, peplomycin, puromycin, streptozocin, ubenimex, zinostatin, denopterin, pteropterin, trimetrexate, 6-mercaptopurine, thiamiprine, thioguanine, 6-azauridine, carmofur, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L-asparaginase, pulmozyme, amsacrine, bisantrene, demecolcine, diaziquone, elliptinium acetate, flutamide, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, mitoxantrone, nitracrine, pentostatin, phenamet, 2-ethylhydrazide, procarbazine, razoxane, erlotonib, urethane, vinblastine and vincristine;
The anti-cancer drug containing a tertiary or secondary nitrogen atom is selected from: altretamine, triethylenemelamine, chlorambuci, chlornaphazine, estramustine, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, pipobroman, actinomycin, anthramycin, carzinophilin, actinomycin D, nogalamycin, porfiromycin, puromycin, streptozocin, tubercidin, fludarabine, ancitabine, azacitidine, cytarabine, dideoxyuridine, enocitabine, floxuridine, L-asparaginase, pulmozyme, aldophosphamide glycoside, bestrabucil, diaziquone, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, mitoguazone, mopidamol, nitracrine, pentostatin, phenamet, razoxane, spirogermanium, tamoxifen, triaziquone, 2,2′,2″-trichlorotriethylamine, vinblastine and vincristine.
Further, the AKR1C3 enzyme-activated anti-cancer prodrug (structural formula II) contains a compound having a structural formula selected from the group consisting of:
Scheme 3: An Assembly for Measuring the Expression Level of AKR1C3 Enzyme
The present invention provides an assembly for measuring the expression level of AKR1C3 enzyme, comprising a reaction measurement component and a control comparison component.
The reaction measurement component is a component that contacts and reacts with the biological sample, and quantitatively or semi-quantitatively associate with the content of PGF2α and/or PGD2 and/or PGH2 in the biological sample according to the signal of the reaction.
This type of component is similar to an ELISA test well plate.
The ELISA test well plate contains a reagent that can react with the target prostaglandin (F2α/D2/H2) and a special color reagent. Quantitation is performed by a photometer based on the depth of the reaction color.
The principle of the test well plate is similar to that of a commercially available ELISA kit. Quantitation is performed by the degree of a microplate reader or a photometer.
Obviously, in order to make the reaction measurement component react with the target prostaglandin accurately and without interference, the measurement component is also equipped with various corresponding auxiliary reagents: a reaction solution, a masking agent solution, a washing buffer solution, a reaction termination solution, and the like.
The control comparison component is a component that is used to compare the signal of the reaction to comparatively compare and obtain the expression level of AKR1C3 enzyme corresponding to the content of PGF2α and/or PGD2 and/or PGH2, the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interference drug, or the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interference drug in the biological sample.
This control comparison component can be a table of known corresponding relationship between concentration and absorbance: a user directly compares the different colors and the corresponding absorbances on the test well plate with the control comparison component and directly obtains the corresponding prostaglandin content level.
After the content level is obtained, the corresponding expression level of AKR1C3 enzyme can be directly obtained (Association Rule 1):
when the content of PGF2α is in the range of A1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content of PGF2α is in the range of B1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content of PGF2α is in the range of C1, the expression level of AKR1C3 enzyme in the biological sample is low;
and/or
when the content of PGD2 and/or PGH2 is in the range of A2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content of PGD2 and/or PGH2 is in the range of B2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content of PGD2 and/or PGH2 is in the range of C2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of A1 is greater than or equal to the maximum value in the range of B1, and the minimum value in the range of B1 is greater than or equal to the maximum value in the range of C1,
the minimum value in the range of A2 is greater than or equal to the maximum value in the range of B2, and the minimum value in the range of B2 is greater than or equal to the maximum value in the range of C2.
Alternatively, the corresponding prostaglandin content grade is obtained after application of the interference drug, and the two are calculated to obtain the content grade difference (the content grade difference corresponds to the content difference within a range, which actually obtains a difference result of a semi-quantitative content) and the corresponding expression level of AKR1C3 enzyme can be obtained according to the following association (Association Rule two):
when the content change of PGF2α is in the range of α1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content change of PGF2α and/or PGD2 and/or PGH2 is in the range of b1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change of PGF2α and/or PGD2 and/or PGH2 is in the range of c1, the expression level of AKR1C3 enzyme in the biological sample is low;
and/or
when the content change of PGD2 and/or PGH2 is in the range of α2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content change of PGD2 and/or PGH2 is in the range of b2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change of PGD2 and/or PGH2 is in the range of c2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of α1 is greater than or equal to the maximum value in the range of b1, and the minimum value in the range of b1 is greater than or equal to the maximum value in the range of c1,
the minimum value in the range of α2 is greater than or equal to the maximum value in the range of b2, and the minimum value in the range of b2 is greater than or equal to the maximum value in the range of c2.
This control comparison component can be a corresponding relationship table: a user uses a photometer or a microplate reader to test the fully reacted and treated test well plate and read it, and then obtains the corresponding prostaglandin content in the sample according to the reading versus a standard degree-content (concentration content, pg/ml concentration for human) curve. With the similar principle and operation as above, the more accurate content change rate can be used to determine the expression level of AKR1C3 enzyme (Association Rule III):
when the content change rate of PGF2α is in the range of α1, the expression level of AKR1C3 enzyme in the biological sample is high;
when the content change rate of PGF2α is in the range of β1, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change rate of PGF2α is in the range of γ1, the expression level of AKR1C3 enzyme in the biological sample is low,
and/or
when the content change rate of PGD2 and/or PGH2 is in the range of α2, the expression level of AKR1C3 enzyme in the biological sample is low;
when the content change rate of PGD2 and/or PGH2 is in the range of β2, the expression level of AKR1C3 enzyme in the biological sample is medium;
when the content change rate of PGD2 and/or PGH2 is in the range of γ2, the expression level of AKR1C3 enzyme in the biological sample is high,
wherein the minimum value in the range of α1 is greater than or equal to the maximum value in the range of β1, and the minimum value in the range of β1 is greater than or equal to the maximum value in the range of γ1,
the minimum value in the range of α2 is greater than or equal to the maximum value in the range of β2, and the minimum value in the range of β2 is greater than or equal to the maximum value in the range of γ2.
It is known to those skilled in the art that although the above description is only based on the content level, it is also feasible to use the photometer or the microplate reader to directly read the test well plate to obtain the corresponding prostaglandin content, and then use Association Rule 1 or Rule 2 to determine, or it is also feasible to use Rule 2 or Rule 3 according to the content change value and content change rate to determine. The above description is not limited to a certain corresponding limit.
Obviously, the above method is only one of the methods for measuring prostaglandins. In fact, reaction measurement components of different formulations corresponding to PGF2α, PGD2, and PGH2 can be used for quantitatively or semi-quantitatively measuring different prostaglandin contents. Then different corresponding relationship tables are correspondingly established to respectively measure the contents of different prostaglandins, the content change and the content change rate before and after the application of the interference drug, so that the test component can be applied to a wider population, and the test result is more accurate and credible.
Scheme 4: Administration Device
The present invention provides an administration device, comprising:
the assembly for measuring the expression level of AKR1C3 enzyme as described above;
An administration assembly, which contains an AKR1C3 enzyme-activated anti-cancer prodrugs.
Obviously, the administration device is equipped with a further administration assembly on the basis of the above-mentioned test assembly: including an administration tool and a drug.
For example, the administration tool can be a injector, and the corresponding drug is a powder for injection (lyophilized or aseptic packing) and a diluted solution for injection; or the injector and the drug are designed as an integrated structure, which is directly injected for administration without further operations of dilution or dissolution.
The administration tool can be a hypospray, and the corresponding drug is a powder for injection (lyophilized or aseptic packing), i. e. a so-called “needle-free injection system”, which relies on propulsion of compressed gas or explosion to inject the powder through the skin into the blood vessels.
The present invention will be described below with reference to specific examples. Those skilled in the art could understand that these examples are only used for describing the invention and do not in any way limit its scope.
The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials of the drugs, the reagents and the like used in the following examples are all commercially available products unless otherwise specified.
The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from the definitions of the terms as generally understood in the art.
All numerical designations, e.g., pH, temperature, time, concentration, and weight, including ranges of each thereof, are approximations that typically may be varied (+) or (−) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”. Reagents described herein are exemplary and equivalents of such may be known in the art.
“Cx-Cy” or “Cx-y” before a group refers to a range of the number of carbon atoms that are present in that group. For example, C1-C6 alkyl refers to an alkyl group having at least 1 and up to 6 carbon atoms.
“Alkoxy” refers to —O-Alkyl.
“Amino” refers to NRpRq wherein Rp and Rq independently are hydrogen or C1-C6 alkyl, or Rp and Rq together with the nitrogen atom to which they are bonded form a 4-15 membered heterocycle.
“Aryl” refers to an aromatic group having carbon atoms and containing no ring heteroatoms and having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). For multiple ring systems, including fused, bridged, and spiro ring systems having aromatic and non-aromatic rings that have no ring heteroatoms, the term “aryl” or “Ar” applies when the point of attachment is at an aromatic carbon atom (e.g., 5,6,7,8 tetrahydronaphthalene-2-yl is an aryl group, as its point of attachment is at the 2-position of the aromatic phenyl ring).
According to specific embodiments of the present application, C6-C10 aryl can be phenyl, naphthyl and various substituted phenyl or naphthyl.
“Heteroaryl” (Heterocyclic aryl) refers to an aromatic group of from 1 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur and includes single ring (e.g. imidazolyl-2-yl and imidazol-5-yl) and multiple ring systems (e.g. imidazopyridyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl). For multiple ring systems, including fused, bridged, and spiro ring systems having aromatic and non-aromatic rings, the term “heteroaryl” applies if there is at least one ring heteroatom, and the point of attachment is at an atom of an aromatic ring (e.g. 1,2,3,4-tetrahydroquinolin-6-yl and 5,6,7,8-tetrahydroquinolin-3-yl). In some embodiments, the nitrogen and/or the sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. The term heteroaryl or 5-15 membered heteroaryl includes, but is not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazopyridyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl.
“Alkyl” refers to a monovalent saturated aliphatic hydrocarbyl group having carbon atom(s) and, in some embodiments, from 1 to 6 carbon atoms. “Cx-y alkyl” refers to an alkyl group having from x to y carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3—), ethyl (CH3CH2—), n-propyl (CH3CH2CH2—), isopropyl ((CH3)2CH—), n-butyl (CH3CH2CH2CH2—), isobutyl ((CH3)2CHCH2—), sec-butyl ((CH3)(CH3CH2)CH—), tert-butyl ((CH3)3C—), n-pentyl (CH3CH2CH2CH2CH2—), and neopentyl ((CH3)3CCH2—).
“Cycloalkyl” refers to a saturated or partially saturated cyclic group with more than 3 carbon atoms and no ring heteroatoms and having a single ring or multiple rings including fused, bridged, and spiro ring systems. For multiple ring systems having aromatic and non-aromatic rings that have no ring heteroatoms, the term “cycloalkyl” applies when the point of attachment is at a non-aromatic carbon atom (e.g. 5,6,7,8-tetrahydronaphthalene-5-yl). The term “cycloalkyl” or “C3-C8 cycloalkyl” includes cycloalkenyl groups. Examples of cycloalkyl groups or C3-C8 cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl.
“Heterocyclic” or “heterocycle” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated or partially saturated cyclic group having carbon atom(s) and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen and includes single ring and multiple ring systems including fused, bridged, and spiro ring systems. For multiple ring systems having aromatic and/or non-aromatic rings, term “heterocyclic”, or “heterocycle”, or “heterocycloalkyl”, or “heterocyclyl” applies when there is at least one ring heteroatom, and the point of attachment is at an atom of a non-aromatic ring (e.g. 1,2,3,4-tetrahydroquinoline-3-yl, 5,6,7,8-tetrahydroquinoline-6-yl, and decahydroquinolin-6-yl). In some embodiments, the heterocyclic groups herein are 3-15 membered, 4-14 membered, 5-13 membered, 7-12 membered, or 5-7 membered heterocycles. In some other embodiments, the heterocycles contain 4 heteroatoms. In some other embodiments, the heterocycles contain 3 heteroatoms. In another embodiment, the heterocycles contain up to 2 heteroatoms. In some embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, sulfinyl, or sulfonyl moieties. Heterocyclyl includes, but is not limited to, tetrahydropyranyl, piperidinyl, N-methylpiperidin-3-yl, piperazinyl, N-methylpyrrolidin-3-yl, 3-pyrrolidinyl, 2-pyrrolidon-1-yl, morpholinyl, and pyrrolidinyl. A prefix indicating the number of carbon atoms (e.g., C3-10) refers to the total number of carbon atoms in the portion of the heterocyclyl group exclusive of the number of heteroatoms. A divalent heterocyclic group will have the appropriately adjusted hydrogen content.
“Ether” refers to a C1-C6 alkyl group substituted with 1-3 C1-C6 alkoxy groups, wherein alkoxy refers to —O-alkyl.
“Halo” or “Halogen” refers to one or more of fluoro, chloro, bromo, and iodo.
“Alkenyl” refers to a linear or branched hydrocarbyl group having carbon atoms and in some embodiments from 2 to 6 carbon atoms or 2 to 4 carbon atoms and having at least 1 site of vinyl unsaturation (>C═<). For example, Cx-y alkenyl refers to an alkenyl group having from x to y carbon atoms and is meant to include, for example, ethenyl, propenyl, 1,3-butadienyl, and the like.
“Alkynyl” refers to a linear monovalent hydrocarbon group or a branched monovalent hydrocarbon group having more than 2 carbon atoms and in some embodiments from 2 to 6 carbon atoms or 2 to 4 carbon atoms and containing at least one triple bond. The term “alkynyl” is also meant to include those hydrocarbyl groups having one triple bond and one double bond. For example, C2-6 alkynyl includes ethynyl, propynyl, and the like.
“Phosphoramidate alkylating agent” refers to an alkylating agent comprising one or more Z5—X5—Y5 moieties bonded to an —O—P(Z1) moiety, where Z5 is a heteroatom such as nitrogen, sulfur or oxygen, X5 is optionally substituted ethylene, Y5 is halo or another leaving group, or Z5—X5—Y5 together form an aziridinyl (NCH2CH2) moiety, and Z1 is defined as above. Such an alkylating agent can react with DNA or another nucleic acid or protein. In some instances, an alkylating agent can be cross-linked with DNA.
The group may be substituted with one or more substituents, e.g., 1, 2, 3, 4 or 5 substituents. Preferably, the substituents are selected from the group consisting of oxo, halo, —CN, NO2, —N2+, —CO2R100, —OR100, —SR100, —SOR100, —SO2R100, —NR100, —SO2R100, —NR101R102, —CONR101R102, —SO2NR101R102, C1-C6 alkyl, C1-C6 alkoxy, —CR100═C(R100)2, —CCR100, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C6-C12 aryl and C2-C12 heteroaryl, or a divalent substituent such as —O—(CH2)—O—, —O—(CH2)2—O—, and, 1-4 substituted methyl groups, wherein each R100, R101, and R102 independently is hydrogen or C1-C8 alkyl; C3-C12 cycloalkyl; C3-C10 heterocyclyl; C6-C12 aryl; or C2-C12 heteroaryl; or R100 and R102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocycle; wherein each alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 halo, 1-3 C1-C6 alkyl, 1-3 C1-C6 haloalkyl or 1-3 C1-C6 alkoxy groups. Preferably, the substituents are selected from the group consisting of chloro, fluoro, —OCH3, methyl, ethyl, iso-propyl, cyclopropyl, —CO2H and salts and C1-C6 alkyl esters thereof, CONMe2, CONHMe, CONH2, —SO2Me, —SO2NH2, —SO2NMe2, —SO2NHMe, —NHSO2Me, —NHSO2CF3, —NHSO2CH2C1, —NH2, —OCF3, —CF3 and —OCHF2.
“Alkylene” refers to a divalent saturated aliphatic hydrocarbyl group having carbon atom(s) and, in some embodiments, from 1 to 6 carbon atoms, and an alkyl group further losing one H atom. “Cu-v alkylene” refers to alkylene groups having from u to v carbon atoms. The alkylene group includes branched and straight chain hydrocarbyl groups. For example, “C1-6 alkylene” includes methylene, ethylene, propylene, 2-methylpropylene, pentylene, and the like.
“Heteroalkylene” refers to an alkylene wherein a chain carbon atom is replaced with a heteroatom such as O, S, N, or P, or a heteroatom containing a substituent.
The “drug” regarding D herein includes without limitation, gemcitibine, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, trimethylmelamine, chlorambucil, chlomaphazine, estramustine, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carubicin, carzinophilin, chromomycin, actinomycin D, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, defofamide, demecolcine, diaziquone, elfomithine, elliptinium acetate, etoglucid, flutamide, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, erlotonib, teniposide, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, urethane, vinblastine, and vincristine.
“Administering” or “administration of” a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administered to a patient by a medical professional or may be self-administered, and/or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is to administer the drug to the patient.
“Cancer” refers to leukemias, lymphomas, carcinomas, and other malignant tumors, including solid tumors, of potentially unlimited growth that can expand locally by invasion and spread throughout the body by metastasis. Examples of cancers include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. Certain other examples of cancers include, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, poor cervical intraepithelial differentiation and in situ carcinoma, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforma, hairy-cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary epithelial carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteo sarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythermia vera, primary brain tumor, small-cell lung tumor, squamous cell carcinoma of both ulcerating and papillary type, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, reticulum cell sarcoma, and Wilm's tumor.
“Patient” and “subject” are used interchangeably to refer to a mammal in need of treatment for cancer. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain embodiments, a “patient” or “subject” may refer to a non-human mammal used in screening, characterizing, and evaluating drugs and therapies, such as, a non-human primate, a dog, cat, rabbit, pig, mouse or a rat.
“Prodrug” refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is made such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.
The patient in the present application refers to a patient suffering from a disease or disorder, complication associated with the AKR1C3 enzyme and its corresponding gene, or further defined as a cancer or tumor corresponding to the cytotoxic prodrug activated by the AKR1C3 enzyme or disorder or cell proliferative disease caused by the cancer or tumor.
“Solid tumor” refers to solid tumors including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph node, pancreas, prostate, skin and soft tissue (sarcoma).
“Therapeutically effective amount” of a drug refers to an amount of a drug that, when administered to a patient with cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer in the patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
“Treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms of cancer; diminishment of extent of disease; delay or slowing of disease progression; alleviation, palliation, or stabilization of the disease state; or other beneficial results. Treatment of cancer may, in some cases, result in partial response or stable disease.
“Tumor cells” refers to tumor cells of any appropriate species, e.g., mammalian such as murine, canine, feline, equine or human.
The above description of embodiments of the present invention does not limit the present invention. Those skilled in the art can make various modifications and changes according to the present invention, and any modification and change within the spirit of the present invention shall be covered in the scope of the claims appended to the present invention.
Since the present invention is based on the following three invention applications: Application No. PCT/US2016/021581, publication No. WO2016/145092 corresponding to Chinese application No. 2016800150788, publication No. Application No. PCT/US2016/025665, publication No. WO2016/061342 corresponding to Chinese application No. 2016800200132, publication No. CN108136214A; and
Application No. PCT/US2016/062114, publication No. WO2017/087428 corresponding to Chinese application No. 2016800446081, publication No. CN108290911A, the above-mentioned three applications are incorporated into the text of the present application for this purpose.
Further, the AKR1C3 inhibitors mentioned in the present invention are for this purpose also incorporated into the present application by reference to the following applications:
With regard to the AKR1C3 inhibitors mentioned in the present invention, it has been reported in the following literatures (Higaki, Y, Usami, et al. Selective and potent inhibitors of human 20a-hydroxysteroid dehydrogenase (AKR1C1) that metabolizes neurosteroids derived from progesterone[J]. CHEMICOBIOLOGICAL INTERACTIONS, 2003, 503-513; Bydal P, Luu—The V, Labrie F, et al. Steroidal lactones as inhibitors of 17beta-hydroxysteroid dehydrogenase type 5: chemical synthesis, enzyme inhibitory activity, and assessment of estrogenic and androgenic activities. [J]. European Journal of Medicinal Chemistry, 2009, 44(2):632-644; Skarydova L, Wsol V, Zivna L, et al. AKR1C3 as a potential target for the inhibitory effect of dietary flavonoids[J]. Chemico-biological interactions, 2010, 178:138-144; Byrns M C, Steckelbroeck S, Penning T M. An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies. [J]. Biochemical Pharmacology, 2008, 75(2):484-493; Bauman, D. R. Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo-keto reductase isoforms: potential antineoplastic agents that work independently of cyclooxygenase isozymes.[J]. Molecular Pharmacology, 2005, 67(1):60-68; Penning, T, M, et al. Inhibition of a major NAD(P)-linked oxidoreductase from rat liver cytosol by steroidal and nonsteroidal anti-inflammatory agents and by prostaglandins.[J]. Proceedings of the National Academy of Sciences, 1983, 8:4504-4508; Davies N J, Hayden R E, Simpson P J, et al. AKR1C isoforms represent a novel cellular target for jasmonates alongside their mitochondrial-mediated effects.[J]. Cancer Research, 2009, 69(11):4769-75; Brozic P, Golob B, Gomboc N, et al. Cinnamic acids as new inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (AKR1C3).[J]. Molecular & Cellular Endocrinology, 2006, 248(1-2):233-235; Yining Zhao, Xuehua Zheng, Hong Zhang, et al. In vitro inhibition of AKR1Cs by sulphonylureas and the structural basis.[J]. Chemico-Biological Interactions, 2015, 240:310-315) that some commercially available drugs have an inhibitory effect on AKR1C3, and these drugs are also inhibitors of the AKR1C3 enzyme:
Glycyrrhetinic acid, Glycyrrhizinate and salt or glycoside thereof, Ursodeoxycholic acid, medroxyprogesterone acetate (MPA), Estradiol, Hexestrol, Bethamethasone, cortisone, Prednisone, methylprednisolone, triamcinolone, Hydrocortisone, Dexamethasone, Spironolactone, Brotizolam, Estazolam, Flunitrazepam, Flurazepam, meclonazepan, Lormetazepam, Midazolam, nimetazepam, Nitrazepam, Temazepam, triazolam, alprazolam, Bromazepam, Chlordiazepoxide, clobazam, clonazepam, Delorazepam, diazepam, fludiazepam, Halazepam, Lorazepam, Medazepam, nordazepam, oxazepam, Prazepam, Cloxazolam, Phenolphthalein, Ipriflavone, Flavoxate Hydrochloride, Purified Micronised Flavonoid Fraction, Seabuckthorn flavone, silibinin, efloxate, Quercetin, Luteolin, Aspirin, Sodium salicylate, Paracetamol, Naproxen, Nabumetone, Diclofenac, Ibuprofen, rofecoxib, Celecoxib, Aceclofenac, Diflunisal, Etodolac, Fenoprofen, Flurbiprofen, ketoprofen, suprofen, Tiaprofenic Acid, Ketorolac, Zomepirac, Mefenamic acid, Flufenamic acid, Meclofenamic Acid, Meloxicam, Oxaprozin, Piroxicam, Tenoxicam, Lornoxicam, Sasapyrine, Sulindac, Tolmetin, Phenacetin, Loxoprofen Sodium, Aminopyrine, Metamizole Sodium, Sudoxicam, Phenylbutazone, Oxyphenbutazone, Chlorpropamide, Tolbutamide, Gliclazide, Glibenclamide, Gliquidone, Glipizide, and Glimepiride.
The following are specific tests and examples of the present invention.
The following tests will disclose experimental data of the applicant for proving relevant conclusions and relevant experimental facts of the present application, and the applicant hereby declares that the rights of the following experimental data belong to the applicant.
The compound
(i.e., the racemic isomer of compound AST-3424, the S-configuration of which is AST-3424
has AKR1C3 enzyme inhibitory activity.
In Vitro Activity Inhibition Experiment of the Compounds on AKR1C3
Experimental apparatus: Waters Acquity I Class UPLC Ultra Performance Liquid Chromatograph equipped with a Xevo G2-XS Q T of HRMS Quadrupole Time-of-flight High-Resolution Mass Spectrometer
Buffers and Materials:
1. PBS phosphate buffered saline solution,
2. PBS phosphate buffered saline solution of 20 mM NADPH
3. PBS phosphate buffered saline solution of 250 μg/mL AKR1C3
4. 50% MeOH/H2O solution of 250 μM AST-3424
5. 50% MeOH/H2O solution of 250 μM progesterone
6. 100% acetonitrile solution of 1 μg/mL propranolol
Step 1, the reaction mixtures were put into Eppendorf tubes (microcentrifuge tubes) in quadruplicate (n=4) according to the table below and mixed gently.
Step 2, the above mixtures were pre-incubated in duplicate at 37° C. for 30 minutes and 60 minutes.
Step 3, another 10 μL of PBS phosphate buffered saline solution of 20 mM NADPH and 2 μL of 50% MeOH/H2O solution of 250 μM progesterone were added to each Eppendorf tube and mixed gently.
Step 4, 50 μL of the mixture in the above step was immediately transferred to 100 μL of 100% acetonitrile solution of 1 μg/mL propranolol (internal standard (IS)).
Step 5, the remaining samples were incubated at 37° C. for 30 minutes, and 100 μL of 100% acetonitrile solution of 1 μg/mL propranolol (internal standard (IS)) was added.
Step 6, 100 μL of reagent water was added to all the samples, vortexed at 1,100 rpm for 5 minutes, and centrifuged at 15,000 rpm for 10 minutes at room temperature.
Step 7, all the samples were loaded on LC/MS to determine the content of reduced progesterone, namely 20α-dihydroprogesterone.
The test conditions for the LC-MS apparatus are shown below.
The gradient of the liquid phase elution
Parameters of the Quadrupole Time-of-flight Mass Spectrometer
Step 9, the reduced progesterone (20α-dihydroprogesterone) is calculated: the peak area of the reduced progesterone, namely 20α-dihydroprogesterone and propranolol, in each sample was determined by LC/MS. The peak area ratios of reduced progesterone to propranolol (i.e., the ratios in the above table) were calculated, and when the time is 0 the ratio is set to 0%.
AKR1C3 activity (%)=[(the amount of reduced progesterone after normalization of the sample) 30 min−(the amount of reduced progesterone after normalization of the sample) 0 min]/[(the amount of reduced progesterone after normalization of the negative control group) 30 min−(the amount of reduced progesterone after normalization of the negative control group) 0 min]*100.
The AKR1C3 activity results in the above table were calculated according to the above formula.
Analysis and Summary of the Experimental Results
Table: The effect of AST-3424 on AKR1C3 activity
The activity was 92.4% when the test value of indometacin was at a concentration of 5 um/L.
The effect of AST-3424 on the production of reduced progesterone: The aforementioned in vitro experiment proves that after pre-incubation for 30 minutes and 60 minutes, AST-3424 at a concentration of 25 μM basically inhibited AKR1C3 activity: compared with the negative control groups, the production of the reduced progesterone, namely 20α-dihydroprogesterone, was reduced to 3.9% and 9.2%, respectively, proving that the compound AST-3424 are inhibitors of AKR1C3 enzyme.
Content Change Experiment of Prostaglandin in Blood of Cynomolgus Monkeys Before and After Administration of AKR1C3 Inhibitor AST-3424
An experiment with three cynomolgus monkeys was performed according to the table below.
Four male cynomolgus monkeys were purchased from Guangxi Xiongsen Primate Development and Experiment Co., Ltd., and all of them were healthy cynomolgus monkeys that had passed the physical examination and have no abnormalities. Among them, three cynomolgus monkeys were used in the experiment of administration and the remaining one was used for preparing blank plasma.
Before administration, and 6, 24, 48 and 72 hours after the start of administration, 1 mL of blood was collected from the femoral vein or other suitable vein, and placed in a blood collection tube without anticoagulant. After collected, the blood sample was placed on ice and allowed to stand for 30-60 minutes before being centrifuged at 3,500 rpm for 10 minutes at 2-8° C. to separate the serum. The collected serum was stored at −80° C. before being analyzed.
Prostaglandin F2 in serum samples was analyzed by conventional ELISA methods. The measurement results are as follows.
The Concentrations of Prostaglandins E2 and F2 in the Serum after Single Administration to Cynomolgus Monkeys by Intravenous Infusion
Six hours after administration of 0.58 mg/kg of AST-3424 to cynomolgus monkeys, the content of prostaglandin F2a in all three cynomolgus monkeys decreased, indicating that the process of prostaglandin F2a secretion in cynomolgus monkeys can be inhibited after administration of AST-3424.
Obviously, it can be found that:
I: the levels of the content of prostaglandin F2a in blood of the three cynomolgus monkeys before using the interfering drugs have a significant difference, which were very different from each other.
II: Further, exploration of the content change of prostaglandin F2a in the three cynomolgus monkeys (101/102/103) after using infectious drugs shows that the contents of prostaglandin F2a of them are decreased within 6 hours while they are decreased or increased at 24, 48 and 72 hours after using AKR1C3 inhibitor (AST-3424), and this experimental fact shows that using an interfering drug (inhibitor) against AKR1C3 enzyme activity should be followed by the detection of change of prostaglandin within a suitable period of time, and for the three cynomolgus monkeys used in this experiment, 6 hours were appropriate.
III: Further, a comprehensive I/II exploration of the three cynomolgus monkeys for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the absolute values of the content change of prostaglandin F2a were also completely different: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change values of prostaglandin F2a levels in their blood after administration of the interfering drug.
IV: Further, a comprehensive I/II exploration of the three cynomolgus monkeys for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the change ratios of the content change of prostaglandin F2a were also completely different: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change ratios of the content of prostaglandin F2a levels in their blood after administration of the interfering drug.
In conclusion, a comprehensive exploration of the three cynomolgus monkeys without administration of interfering drugs and for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the absolute value/relative change ratios of the content change of prostaglandin F2a was positively correlated with PGF2α in cynomolgus monkeys without administration of interfering drug: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change value/relative change ratio of the content of prostaglandin F2a levels in their blood after administration of the interfering drug.
In fact, after using traditional immunohistochemical staining method (IHC) to detect AKR1C3 in the liver tissues obtained from these three Cynomolgus monkeys, it was found that the staining area of No.s 101 and 103 cynomolgus monkeys was higher than that of No. 102 Cynomolgus monkey, i.e. the expression level of AKR1C3 enzyme in No. 101 and 103 Cynomolgus monkeys was higher than that of No. 102 Cynomolgus monkey.
The above in vitro and in vivo experiments confirm that route 1/2/3 is truly present, the conclusion by measuring the content of PGF2α and/or PGD2 and/or PGH2 or the content change and content change ratio before and after administration of interfering drugs in vitro and then combining with the data statistics can correlate the expression levels of AKR1C3 enzyme in biological samples, that is, it is clinically possible to directly obtain the expression levels of AKR1C3 enzyme of patients by measuring the content of prostaglandin, thereby screening a cancer or tumor patient with an appropriate expression level of AKR1C3 enzyme and administering the AKR1C3 enzyme-activated anti-cancer prodrug disclosed in the present invention to the patient.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911219742.2 | Dec 2019 | CN | national |
The present invention, belonging to the field of research and development of formulation of anti-cancer compounds, relates to the research and development of injection solutions of compounds disclosed in Patent Application No. PCT/US2016/021581 (Publication No. WO2016145092A), which corresponds to Chinese Patent Application No. 2016800150788 (Publication No. CN107530556A); PCT Patent Application No. PCT/US2016/062114 (Publication No. WO2017087428A1), which corresponds to Chinese Patent Application No. 2016800446081 (Publication No. CN108290911A); and PCT Patent Application No. PCT/US2016/025665 (Publication No. WO2016/161342), which corresponds to Chinese Patent Application No. 2016800200132 (Publication No. CN108136214A).
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/133538 | 12/3/2020 | WO |
